CN110878369A - Kit for detecting nucleic acid of neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology and application thereof - Google Patents
Kit for detecting nucleic acid of neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting nucleic acid of neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology and application thereof. The kit realizes the amplification of pathogen nucleic acid fragments through the steps of cracking collected samples by cell lysate to release pathogen nucleic acid, and then performing reverse transcription and transcription under the action of reverse transcriptase and T7RNA polymerase. The amplified RNA product is identified and captured by a specific probe in the detection liquid to form an RNA amplification product-specific probe-gold probe compound, and the compound is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of pathogen nucleic acid is realized. The method has no RNA extraction process, does not need special instruments, is based on RNA isothermal amplification, is not easy to pollute in actual detection, has the advantages of high sensitivity, strong specificity and simple operation, and makes the wide application of the Neisseria gonorrhoeae nucleic acid detection possible.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for detecting nucleic acid of neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology and application thereof.
Background
Neisseria Gonorrhoeae (NG) is a pathogen causing gonorrhea, is a strict human parasitic bacterium, is often present in cells of purulent secretions of acute urethritis and vaginitis, and is in an oval or bean shape, and the length of the thallus is 0.6-0.8 mu m, and the width of the thallus is about 0.5 mu m. Often arranged in pairs with adjacent faces flattened or slightly concave, like two beans paired together. No flagella, no sporulation and gram negative staining.
Neisseria gonorrhoeae is a pathogenic bacterium of gonorrhea, has strict human parasitism, and has strong adaptability and invasiveness to human bodies. Gonorrhea is one of the infectious diseases with the highest incidence in developing countries and is also the venereal disease with the highest incidence in China at present. In the early stage of neisseria gonorrhoeae infection, a human body does not have clinical symptoms, but serious genitourinary tract diseases can be caused if the neisseria gonorrhoeae infection cannot be diagnosed in time, particularly, pelvic inflammation or secondary infertility is often caused by female patients, and the timely and accurate diagnosis of neisseria gonorrhoeae infection becomes the key for treating gonorrhoea. Gonococcal infections can cause purulent infections of the urogenital system, and can also cause infections outside the urogenital tract, such as proctitis, pharyngitis, neonatal conjunctivitis, and the like. The clinical manifestations are urethritis and cervicitis, and typical symptoms are dysuria, frequent micturition, urgent micturition, odynuria, mucus or purulent secretion and the like.
Gonorrhea caused by neisseria gonorrhoeae infection is a common sexually transmitted disease, and particularly has a high incidence rate in people in China.
The method for detecting NG infection in an early, rapid and accurate manner is of great significance. In combination with domestic conditions, in the experimental diagnosis of NG infection of genitourinary tracts, although a classical cell culture method is specific, the method has long time consumption, high cost and high requirements on technical equipment, is not suitable for treating a large number of samples and has low sensitivity; the PCR method can directly detect NG nucleic acid, has high sensitivity, strong specificity and higher detection speed, has considerable advantages in shortening the detection window period and improving the pathogen detection rate, and is one of the main methods for detecting NG pathogens. The existing commonly used neisseria gonorrhoeae nucleic acid detection is based on a fluorescence PCR method, the methods need a complex RNA extraction process, special PCR amplification conditions, a special laboratory and a fluorescence quantitative PCR instrument, and pollution is very easy to generate in the detection process, so that the wide application of the methods is limited, and the methods are not beneficial to popularization and application in some communities and remote hospitals. Therefore, there is still a need to find a method for detecting NG nucleic acid, which is simple, rapid, inexpensive, and highly sensitive. The invention combines the RNA constant temperature amplification technology and the colloidal gold chromatography technology, and can realize the detection of the nucleic acid of the neisseria gonorrhoeae in a shorter time.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a kit for detecting nucleic acid of neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology and application thereof. The kit realizes the amplification of pathogen nucleic acid fragments through the steps of cracking collected samples by cell lysate to release pathogen nucleic acid, and then performing reverse transcription and transcription under the action of reverse transcriptase and T7RNA polymerase. The amplified RNA product is identified and captured by a specific probe in the detection liquid to form an RNA amplification product-specific probe-gold probe compound, and the compound is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of pathogen nucleic acid is realized. Therefore, the invention has no complex RNA extraction process, does not need special instruments, is not easy to pollute in actual detection based on the characteristic that RNA molecules are easy to degrade, has the advantages of high sensitivity, strong specificity and simple operation, and makes the wide application of the Neisseria gonorrhoeae nucleic acid detection possible.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, a kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology is provided, which comprises:
1) amplification reaction solution: containing 40mM Tris-HCl (pH 8.0), 12mM MgCl270mM KCl, 15% DMSO, 5mM DTT, 1mM each dNTP, 2mM each NTP, 0.2. mu.M amplification primers, two pairs of primers required for amplification: neisseria gonorrhoeae amplification primers and endoplasmic control 18S primers;
(1) neisseria gonorrhoeae (a section of a conserved region sequence of the PorB gene) amplification primer:
NG-R primer (5 '-3'): TAATCTGACTCACTAACGGGAGACTCAGACTG CGGACTGATCG, respectively;
NG-F primer (5 '-3'): TTGGTCGACGTACAAACTAGC, respectively;
(2) amplification primers for internal reference gene (a conserved region sequence of human 18 SrRNA):
internal reference-R primer (5 '-3'):
TAATACGACTCACTATAGGGAGACACAGTTATCCAAGTAGGAG；
internal reference-F primer (5 '-3'): TCCTGCCAGTAGCATATGCT, respectively;
when designing the primers, the high amplification efficiency of the respective single primers and the interference resistance among different primers are ensured. The 5' ends of the R primers of the two pairs of primers are introduced with a T7RNA polymerase promoter sequence.
2) Amplification enzyme: including reverse transcriptase (e.g., AMV or M-MLV), T7RNA polymerase, and RnaseH.
3) Cell lysate (purchased from Signosis, USA, Cat. CL-0001): the cells can be lysed, releasing the nucleic acids.
4) Detection liquid: contain the nucleic acid probe (gold probe) of colloidal gold granule mark, the specific probe of every index, C line color development probe, every index specific probe has two kinds, is CES series and LES series respectively, and wherein CES series and LES series can design many again, specifically as follows:
(1) neisseria gonorrhoeae specific probes (5 '-3'):
NG-CES1：TACGTCTTGAGAGGGAAttttGCTCGACTTGCCACCGAATA；
NG-LES1：TCGGGCCAAGTGCAATCCttttGCCTCAAAGACGGACGCCTTC T；
NG-LES2：ATCAATGCCTACGATATTttttGCCTCAAAGACGGACGCCTTC T；
(2) internal reference probe (5 '-3')
Intrinsic reference CES 1: TTGGCTGAAGAATCCAACttttATCTGTATAGTGTCTGT, respectively;
intrinsic reference CES 2: TTGACATGGAGCCTGCGGttttATCTGTATAGTGTCTGT, respectively;
internal reference LES 1: CTTAATTTGACTCAACACttttCGCAGTGCTCGAGCTCTGAG C, respectively;
internal reference LES 2: CCTAGAAGCACGTCGTTCGttttCGCAGTGCTCGAGCTCTGA G, respectively;
internal reference LES 3: AATACAGGACTCTTTCttttCCGCAGTGCTCGAGCTCTGAGC, respectively;
(3) c line color probe (5 '-3')
TCAGATCACTATGTACttttCGCAGTGCTCGAGCTCTGAGC；
(4) Gold probe
The 5' end of the gold probe is modified by sulfhydrylation, and the sequence is as follows:
5’-CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGC TCGAGCACTGCG-3’
5) the test paper strip: the test paper strip is fixed on a PVC bottom plate and sequentially comprises a sample pad, an N C film and absorbent paper from left to right; the NC film is provided with a C line (quality control line) and two T lines (detection lines), and the directions from the sample pad to the absorbent paper are NG-T line, internal reference-T line and C line respectively (as shown in figure 3); NG-T coated probe, internal reference-T coated probe and C line coated probe, wherein the specific sequence (5 '-3') is as follows:
NG coating the probe: ACACCAGCTATAGATAttttACACCAGCTATAGATA, respectively;
internal reference coating probe: CAGACACTATACAGATttttCAGACACTATACAGAT, respectively;
c, coating of a probe: GTACATAGTGATCTGAttttGTACATAGTGATCTGA are provided.
The invention provides a method for detecting nucleic acid of neisseria gonorrhoeae by using the kit for detecting nucleic acid of neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology, which comprises the following steps:
(1) isothermal amplification of RNA
The detection indexes of the invention are two: nucleic acid of Neisseria gonorrhoeae and human reference gene. A pair of (F/R primers) amplification primers is designed for each index, wherein the 5' end of the R primer carries a T7RNA polymerase promoter. The invention realizes the amplification of each index nucleic acid in the same amplification tube, and the specific steps are as follows: during amplification, under the action of an R primer with a T7 promoter and reverse transcriptase, converting RNA to be detected into an RNA-cDNA hybrid; RNA in cDNA is digested by RnaseH in the amplified enzyme to obtain single-stranded cDNA; synthesizing a second strand under the action of the F primer and the DNA polymerase function of reverse transcriptase to form double-stranded DNA with a T7 promoter; the double-stranded DNA with the T7 promoter is transcribed to generate an RNA molecule product under the action of T7RNA polymerase. The transcribed RNA molecule product can enter a circulating amplification process, firstly, the F primer is combined with the transcribed RNA molecule product, and the transcribed RNA is converted into an RNA-cDNA hybrid under the action of reverse transcriptase; RNA in cDNA is digested by RnaseH in the amplified enzyme to obtain single-stranded cDNA; the R primer will then bind to the single-stranded cDNA, synthesize a second strand under the action of the DNA polymerase function of the reverse transcriptase, enrich and synthesize more double-stranded DNA molecules with the T7 promoter again, provide more transcription templates for the T7RNA polymerase, and further generate a large amount of RNA molecule products under the action of the T7RNA polymerase (see FIG. 1).
The invention designs the internal quality control gene detection for monitoring the effectiveness of sample collection and the effectiveness of an amplification system. When the sample is qualified, the sample must contain human exfoliated cells and can be detected in the detection process, when the sample is negative, the endoplasmic control is positive, otherwise, the whole detection needs to be re-sampled for retesting.
(2) Gold probe chromatography
a, designing a specific probe, a gold probe, a C-line color probe and a coated probe
Specific probes: each index-specific probe is included in two categories: CES series and LES series, multiple probes can be designed for each probe. The CES probe comprises two parts, one end of the CES probe can be specifically combined with an amplified RNA product, the other end of the CES probe can be assembled with a coated probe coated on an NC membrane to play a role in fixing the amplified product RNA, and the two parts are linked by 4-5T. Each LES probe also comprises two parts, one end of each LES probe can be specifically combined with the amplified RNA product, the other end of each LES probe can be combined with the gold probe to play a role in color development of the linked gold probe, and the two parts are linked by 4-5T.
Gold probe: the 5' end of the gold probe is modified by sulfhydrylation, and a sulfhydryl group can form a covalent bond with the colloidal gold particles and is marked on the colloidal gold particles. The gold probe may be bound to one end of a specific probe LES.
Coating the probe: the coated probe is fixed on an NC membrane and can be combined with one end of a specific probe CES to play a role in fixation. Each coated probe contains two copies, and each copy is connected by 4-5T.
C-line color probe: comprises two parts which are linked by 4-5T. One end of the probe can be combined with a gold probe, and the other end of the probe can be combined with a C-coated probe coated on an NC membrane. During chromatography, the C-line chromogenic probe can form a 'C-line chromogenic probe-gold probe' complex no matter whether RNA amplification products exist or not, and the complex can be captured and intercepted by the C-line on an NC membrane by the probe during chromatography to form a strip visible to naked eyes. The probe can control the quality of the test paper strip and the detection liquid, and the chromatography process is error-free.
The specific probe is designed without crossing between different probes with the same index, and CES series are not crossed with a gold probe and a coated probe, so that the detection specificity is ensured.
The CES series and LES series of the specific probes are designed to improve the efficiency of immobilization and to bind more gold probes, thereby improving the sensitivity of detection.
b, detecting by test paper
The test strip is provided with a detection line and a quality control line, wherein the detection line comprises NG-T and internal reference-T, wherein the NG coated probe coated at the NG-T can be specifically combined with one end of a Neisseria gonorrhoeae CES series probe; the internal reference coated probe coated at the internal reference-T can be specifically combined with one end of the internal reference CES series probe. The C-line coated probe coated on the quality control line (C line) can be specifically combined with the C-line chromogenic probe. And (3) hybridizing the specific probe CES, the specific probe LES, the gold probe and the specific amplification product of the nucleic acid to be detected, and dripping the hybridized product on a test strip for chromatography, wherein the detection line develops color to indicate that the nucleic acid to be detected exists, and the quality control line develops color to indicate that the detection is effective (as shown in figure 2).
The working process of the method of the invention using the kit is introduced as follows by combining the above principle:
(1) nucleic acid extraction
Collecting a urethral swab or vaginal swab sample of a suspected venereal patient, and releasing the virus RNA molecules by using a cell lysate lysis method.
(2) Isothermal amplification of RNA
mu.L of nucleic acid extract was added to 17. mu.L of amplification reaction solution containing Neisseria gonorrhoeae and internal reference primer, heated at 95 ℃ for two minutes, preheated at 42 ℃ for 2 minutes, added with 1. mu.L of amplification enzyme, and amplified at 42 ℃ for 1 hour at constant temperature. If Neisseria gonorrhoeae exists in the sample to be detected, a large amount of amplification enrichment can be carried out on the index RNA molecules during amplification.
(3) Chromatography of test paper strip
a, prehybridization
The RNA isothermal amplification product was mixed with the detection solution (including specific probe, gold probe and C-line color probe) and prehybridized at 42 ℃ for 10 minutes. The amplified RNA molecules are combined with specific probes (including CES series probes and LES series probes) in a complementary pairing mode. One end of CES series probe is hybridized and complemented with RNA molecule, and the other end is combined with coating probe on NC film; one end of the LES series probe is hybridized and complementarily paired with the RNA molecule, the other end can be complementarily paired and combined with the gold probe, and when an amplification product exists, a CES probe-RNA molecule-LES probe-gold probe compound can be formed.
b, chromatographic detection
Dropping the pre-hybridization product at the sample pad of the test strip, carrying out chromatography on the pre-hybridization solution along the NC membrane in the direction of absorbent paper, and when an RNA amplification product to be detected exists, forming a 'CES probe-RNA molecule-LES probe-gold probe complex', and intercepting the complex by a coating probe coated on the NC membrane during chromatography to form a strip which is seen by naked eyes and is positive (as shown in figure 4).
If the RNA product to be detected is not amplified, the CES probe-RNA molecule-LES probe-gold probe complex is not formed, the colloidal gold particles cannot be gathered at the T line, and no visible band is formed, which is negative (as shown in FIG. 4).
The C-line chromogenic probe can form a 'C-line chromogenic probe-gold probe' complex no matter whether the RNA product to be detected is amplified or not, the complex flows forwards along an NC membrane during chromatography, and when the complex reaches the C line, the complex is combined with a sequence coated at the C line, so that the complex is retained at the C line to form a macroscopic colored band, and the experimental result is effective (as shown in figure 4).
In a second aspect, the application of the kit for detecting the nucleic acid of the neisseria gonorrhoeae based on the RNA isothermal amplification-gold probe chromatography technology in preparing a reagent for detecting the neisseria gonorrhoeae is provided.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention can amplify two indexes of neisseria gonorrhoeae and reference gene simultaneously in the same tube by the RNA isothermal amplification method, the amplified nucleic acid product is RNA, the RNA is easy to degrade in natural environment, and compared with the PCR method, the invention can amplify DNA to prevent pollution more easily. RNA isothermal amplification is carried out in an environment of 42 ℃, and amplification reaction can be realized even in a water bath kettle, so that the requirements of experimental instruments are reduced to the maximum extent.
2. Meanwhile, the invention can ensure high amplification efficiency of each single primer, no interference among different primers and good overall amplification effect through multiple tests when designing the primers.
3. The specific probe CES series and the specific probe LES series introduced during design have the function of bridge molecular components, and the amplification probe and the RNA nucleic acid amplification fragment are successfully combined in series by the two probes to realize specific detection of the index RNA nucleic acid fragment. By using the two sets of probes, any one set of probes and the index nucleic acid amplification fragment can not be successfully immobilized on an NC membrane when the hybridization fails, so that a positive detection result cannot be generated, and the detection specificity is ensured. The results of the detection of 20 other microorganisms listed in Table 2 by the kit of the present inventionAre all negative, and prove that the kit of the invention has no cross reaction with other microorganisms. Wherein each set of probe can be designed into more than two sets, and the design is favorable for improving the sensitivity of the test strip. The minimum detection limit of the kit of the invention to the reference substance of the PCR kit for detecting gonorrhea from China food and drug institute is 1 multiplied by 102cfu/mL. The detection sensitivity and specificity of the neisseria gonorrhoeae of 320 clinical samples with diagnosis results related to genital tract infection are higher than those of a commercial fluorescence quantitative PCR kit for detecting neisseria gonorrhoeae.
4. The invention adopts RNA isothermal amplification technology and test strip chromatography technology, not only applies the characteristic of low requirement of RNA isothermal amplification on instruments, but also successfully integrates the characteristic of rapidness of colloidal gold. The test paper strip is used for detecting nucleic acid, and the result can be interpreted only in about 10 min. The method is also very simple in operation, has low technical requirements on experimenters, does not need special instruments and equipment, and is easy to popularize the nucleic acid detection of the neisseria gonorrhoeae to the basic level and remote rural medical institutions.
Drawings
FIG. 1 is a schematic diagram of isothermal amplification of RNA;
FIG. 2 is a schematic diagram of a test strip color development;
FIG. 3 is a schematic view of the test strip;
FIG. 4 is a schematic diagram of detection of negative and positive;
a: NG negative, internal reference negative;
b: NG is negative, and internal reference is positive;
c: NG positive, internal reference positive;
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The experimental methods in the following examples, in which specific conditions are not specified, are generally performed according to conventional conditions, such as "molecular cloning: the conditions described in the laboratory Manual 3 rd edition (New York: Cold Spring Harbor laboratory Press,2005) were carried out.
[ example 1 ] preparation of test paper for nucleic acid detection
The main raw materials required for preparing the nucleic acid detection test strip are as follows: nitrocellulose membranes (NC membranes), sample pads, absorbent paper, PVC base plates, and the like.
1. Film spraying:
detection line NG-T: can capture and bind a NG specific probe CES sequence, 10 mu M NG coated probe, and the film spraying amount is as follows: 2-3 muL/cm;
detecting internal reference-T: the probe can capture and combine a CES sequence of an internal reference specific probe, 10 mu M of an internal reference coated probe, and the film spraying amount is as follows: 2-3 muL/cm;
quality control line (line C): can capture and bind to a C line chromogenic probe sequence, a 10 mu M C line coated probe, a film spraying amount: 2-3 muL/cm;
after the film spraying is finished, the film is automatically crosslinked once in an ultraviolet crosslinking instrument, and the film is placed in a clean constant temperature cabinet at 37 ℃ for drying for 2 hours and stored in a dry environment for later use.
2. Test strip assembly
And respectively cutting 2cm long absorbent paper, the coated NC film and the sample pad, and sequentially fixing the absorbent paper, the coated NC film and the sample pad on a PVC base plate from top to bottom to obtain the detection test strip. The structure of the test strip is shown in fig. 3.
[ example 2 ] sensitivity test
Gradient dilution is carried out on Neisseria gonorrhoeae (derived from a gonorrhoea PCR kit reference product of China food and drug testing institute) for determining the lowest detection limit, 3-5 parts of virus dilution of each gradient are repeated, 20 times of repeated detection is carried out on each gradient, the level with 90% -95% positive detection rate is taken as the lowest detection limit, and the detection results are as follows:
NG minimum detection limit detection
TABLE 1.1 data of the detection experiments for different Titers of NG
TABLE 1.2 NG minimum detection limit test results
Finally, the detection sensitivity of the kit is determined as follows:
| detecting the index | Minimum limit of detection |
| NG | 1×102cfu/mL |
[ example 3 ] specificity verification
1, test strains
Different microorganisms are detected after nucleic acid is extracted, and the specificity of the primer and probe design of the kit is verified. The relevant pathogens and titers were as follows:
table 2 specificity verification test strain information
2, test results
The test results were as follows:
TABLE 3 results of the specificity verification test
3, conclusion
The data show that the detection results of the kit disclosed by the invention on the microorganisms are negative, so that the kit disclosed by the invention is proved to have no cross reaction with other microorganisms, and the strong specificity of the kit for detecting pathogens is embodied.
Example 4 validation of clinical samples
1, clinical sample information
A total of 320 urine swab or vaginal swab samples from the first hospital in Wuhan City were tested, wherein 189 and 131 male and 131 female specimens, respectively, were used, and the ratio was 59.06% and 40.94%. Of the 452 specimens, patients were 67 years old at the maximum and 16 years old at the minimum, the mean age was 31.25 years old, the standard deviation was 33.5 years old, the median was 29 years old, and the diagnosis of patients in the cohort was associated with genital tract infections.
2, detecting the result
When in detection, the kit and a certain commercial Neisseria gonorrhoeae fluorescence quantitative PCR kit are used for simultaneously detecting samples, and detection results are summarized into a four-table as follows:
the 'gene sequencing method' is adopted for retesting 5 inconsistent samples, the result is positive in 3 samples, the kit is positive in all the samples, the kit is used for detecting negative samples of a commercial Neisseria gonorrhoeae detection fluorescent quantitative PCR kit, the retesting result shows that 3 samples and 2 samples are missed in the commercial Neisseria gonorrhoeae detection fluorescent quantitative PCR kit, and obviously, the kit has higher detection sensitivity and stronger specificity in detecting the Neisseria gonorrhoeae of clinical samples.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400>11
cctagaagca cgtcgttcgt tttcgcagtg ctcgagctct gag 43
<210>12
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
aatacaggac tctttctttt ccgcagtgct cgagctctga gc 42
<210>13
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
tcagatcact atgtactttt cgcagtgctc gagctctgag c 41
<210>14
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
cctactctgc agtgctccat cgtacgtctg tcatttttgc tcagagctcg agcactgcg 59
<210>15
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
acaccagcta tagatatttt acaccagcta tagata 36
<210>16
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
cagacactat acagattttt cagacactat acagat 36
<210>17
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
gtacatagtg atctgatttt gtacatagtg atctga 36
Claims (2)
1. A kit for detecting nucleic acid of Neisseria gonorrhoeae based on RNA isothermal amplification-gold probe chromatography technology is characterized by comprising:
1) amplification reaction solution: containing 40mM Tris-HCl (pH 8.0), 12mM MgCl270mM KCl, 15% DMSO, 5mM DTT, 1mM of each dNTP, 2mM of each NTP, 0.2 mu M of each amplification primer, and two pairs of primers required for amplification: neisseria gonorrhoeae amplification primers and endoplasmic control 18S primers;
(1) n. gonorrhoeae amplification primers:
NG-R primer (5 '-3'): TAATCTGACTCACTAACGGGAGACTCAGACTGCGGACTGATCG, respectively;
NG-F primer (5 '-3'): TTGGTCGACGTACAAACTAGC, respectively;
(2) amplification primers for reference genes:
internal reference-R primer (5 '-3'):
TAATACGACTCACTATAGGGAGACACAGTTATCCAAGTAGGAG；
internal reference-F primer (5 '-3'): TCCTGCCAGTAGCATATGCT, respectively;
2) amplification enzyme: comprises three types, reverse transcriptase, T7RNA polymerase and RnaseH; preferably the reverse transcriptase is AMV or M-MLV;
3) nucleic acid extraction reagent: cell lysate, purchased from Signosis, USA, Cat # CL-0001;
4) detection liquid: contain the nucleic acid probe (gold probe) of colloidal gold granule mark, the specific probe of every index, C line color development probe, every index specific probe has two kinds, is CES series and LES series respectively, and wherein CES series and LES series can design many again, specifically as follows:
(1) neisseria gonorrhoeae specific probes (5 '-3'):
NG-CES1：TACGTCTTGAGAGGGAAttttGCTCGACTTGCCACCGAATA；
NG-LES1：TCGGGCCAAGTGCAATCCttttGCCTCAAAGACGGACGCCTTCT；
NG-LES2：ATCAATGCCTACGATATTttttGCCTCAAAGACGGACGCCTTCT；
(2) internal reference probe (5 '-3')
Intrinsic reference CES 1: TTGGCTGAAGAATCCAACttttATCTGTATAGTGTCTGT, respectively;
intrinsic reference CES 2: TTGACATGGAGCCTGCGGttttATCTGTATAGTGTCTGT, respectively;
internal reference LES 1: CTTAATTTGACTCAACACttttCGCAGTGCTCGAGCTCTGAGC, respectively;
internal reference LES 2: CCTAGAAGCACGTCGTTCGttttCGCAGTGCTCGAGCTCTGAG, respectively;
internal reference LES 3: AATACAGGACTCTTTCttttCCGCAGTGCTCGAGCTCTGAGC, respectively;
(3) c line color probe (5 '-3')
TCAGATCACTATGTACttttCGCAGTGCTCGAGCTCTGAGC；
(4) Gold probe
The 5' end of the gold probe is modified by sulfhydrylation, and the sequence is as follows:
5’-CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGCTCGAGCACTGCG-3’
5) the test paper strip: the test strip is fixed on a PVC bottom plate, and a sample pad, an NC membrane and absorbent paper are arranged from left to right in sequence; the NC film is provided with a C line (quality control line) and two T lines (detection lines), and the directions from the sample pad to the absorbent paper are NG-T line, internal reference-T line and C line respectively (as shown in figure 3); NG-T coated probe, internal reference-T coated probe and C line coated probe, wherein the specific sequence (5 '-3') is as follows:
NG coating the probe: ACACCAGCTATAGATAttttACACCAGCTATAGATA, respectively;
internal reference coating probe: CAGACACTATACAGATttttCAGACACTATACAGAT, respectively;
c, coating of a probe: GTACATAGTGATCTGAttttGTACATAGTGATCTGA are provided.
2. Use of the kit of claim 1 for the preparation of a neisseria gonorrhoeae detection reagent.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201911314374.XA CN110878369B (en) | 2019-12-19 | 2019-12-19 | Kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology and application thereof |
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| CN201911314374.XA CN110878369B (en) | 2019-12-19 | 2019-12-19 | Kit for detecting neisseria gonorrhoeae nucleic acid based on RNA isothermal amplification-gold probe chromatography technology and application thereof |
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Cited By (1)
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| CN111455099A (en) * | 2020-03-24 | 2020-07-28 | 武汉中帜生物科技股份有限公司 | Novel coronavirus (2019-nCoV) nucleic acid detection colloidal gold chromatography kit and application thereof |
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